The approaches described in this section are approaches that could be pursued, but not necessarily approaches that have been previously conceived or pursued. Therefore, unless otherwise indicated, it should not be assumed that any of the approaches described in this section qualify as prior art merely by virtue of their inclusion in this section.
Light sheet microscopy (LSM) is a technique in which an specimen under investigation is illuminated with a sheet of laser light such that the fluorescence emitted from the specimen is recorded in the orthogonal direction with an image detector (such as a CCD, CMOS, sCMOS cameras or equivalent/similar). Optical sections are obtained by moving the object, which can then be combined to obtain a 3D representation of the object.
LSM has wide range of variants that have been developed in recent years, adopting names such that Selective Plane Illumination Microscopy (SPIM), Ultramicroscopy, Digital Scanned Laser Light Sheet Fluorescence Microscopy (DSLM), Orthogonal Plane Fluorescence Optical Sectioning (OPFOS), Thin Laser Light Sheet Microscopy (TLSM), Objective Coupled Planar Illumination (OCPI), among others. (See, e.g., Siedentopf, H. & Zsigmondy, R., Ann. Phys.-Berlin 10, 1-39 (1902); Voie, A. H., et al., J. Microsc.-Oxf. 170, 229-236 (1993); Fuchs, E., et al., Opt. Express 10, 145-154 (2002); Huisken, J., et al., Science 305, 1007-1009 (2004); Holekamp, T. F., et al., Neuron 57, 661-672 (2008); Dodt, H. U. et ai, Nat. Methods A, 331-336 (2007); Huisken, J. & Stainier, D. Y. R., Developments, 1963-1975 (2009); and Keller, P. J. & Stelzer, E. H. K., Curr. Opin. Neurobiol. 18, 624-632 (2008), and patents US2012098949 A1, US 20070109633 A1, EP 2444833 A1 and EP 2494399 A2, the disclosures of each of which is incorporated herein by reference).
All the set-ups for LSM to date come with a number of problems:                The alignment is critical. The distance of the objective to the light-sheet must be set with a precision of microns within the DoF of the objective, and the light sheet must be at 90° from the optical axis of the collection objective.        A change of the collection objective requires realignment and adjustment of the distance between the object and the light sheet.        Changes in the internal refractive index of the specimen effectively change the focal distance of the objective and can induce deformations, curvature and other inhomogeneities of the light sheet. These may result in a loss of focal position and therefore in a blurred image.        To obtain a 3D image                    The object needs to be moved (either in the z direction or rotated).            Both the light sheet and the collection objective must be scanned (displaced) in the z direction, always maintaining constant the distance with the light sheet.                        Both of these induce mechanical movements or pressure waves that may interfere with the specimen and the measurement.        All the above mentioned points prevent the technique to be used for fast 3D imaging of biological specimens. Moreover, for in-vivo imaging the movement can perturb the specimen behaviour and development, resulting in an invasive technique.        
It is desirable for an imaging system to overcome the problems described above.